Serum agglutinating titres against Renibacterium salmoninarum the causative agent of bacterial kidney disease, in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Serum agglutinating titres against Renibacterium salmoninarum were determined from clinically infected, sub-clinically infected and non-infected rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L. Titres ≥16 occurred in clinically infected rainbow trout, but declined after a few months when disease signs were absent. Non-infected rainbow trout had titres ≤ 16. No correlation between the agglutinating titre and the level of infection was found in farmed Atlantic salmon. Variable titres were found in wild salmon broodstock although there was no evidence of BKD during 4 years of testing.     

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.     

Detection of Renibacterium salmoninarum antigen in migrating adult chum salmon (Oncorhynchus keta) in Japan.

Renibacterium salmoninarum antigen was detected in the kidney of migrating chum salmon (Oncorhynchus keta) using the indirect dot blot assay and indirect fluorescent antibody test. The adult chum salmon had migrated into a bay in which cultured coho salmon infected with R. salmoninarum were present. Antigen was detected in 5% of the chum salmon although they did not have clinical signs of bacterial kidney disease (BKD). This report describes the first case of R. salmoninarum antigen detection among wild chum salmon populations in eastern Asia.     

Efficacy of cellular vaccines and genetic adjuvants against bacterial kidney disease in chinook salmon (Oncorhynchus tshawytscha)

DNA adjuvants and whole bacterial cell vaccines against bacterial kidney disease (BKD) were tested in juvenile chinook salmon. Whole cell vaccines of either a nonpathogenic Arthrobacter spp. or an attenuated Renibacterium salmoninarum strain provided limited prophylactic protection against acute intraperitoneal challenge with virulent R. salmoninarum, and the addition of either synthetic oligodeoxynucleotides or purified R. salmoninarum genomic DNA as adjuvants did not increase protection. However, a combination of both whole cell vaccines significantly increased survival among fish naturally infected with R. salmoninarum, and the surviving fish treated with the combination vaccine exhibited reduced levels of bacterial antigens in the kidney. This is the first demonstration of a potential therapeutic effect of a whole cell vaccine against BKD.     

Lack of uptake of Renibacterium salmoninarum by gill epithelia of rainbow trout

The role of rainbow trout ( Oncorhynchus mykiss , Walbaum) gill cells in uptake of the salmonid pathogen Renibacterium salmoninarum was examined using in vitro , whole animal and isolated perfused head models. Uptake of the pathogen was observed only when dissected gill tissue was exposed to the live pathogen. In contrast, live cells of Yersinia ruckeri interacted with the gill epithelia in the isolated perfused head model, confirming the findings of previous studies with whole animal and in vitro systems. These results are discussed in relation to the role of gill tissue in bacterial kidney disease (BKD) pathogenesis and in antigen trapping.     

Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout,coho and Atlantic salmon

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.     

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.     

Use of ELISA to monitor bacterial kidney disease in naturally spawning chinook salmon

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum (Rs) is a serious problem among Pacific Northwest salmon hatcheries and has raised concerns that salmon reared in hatcheries may spread BKD to natural populations. In order to monitor the potential spread of this disease to salmon spawning in nature, a method must be available to collect and analyze tissues from naturally spawning salmon. Kidney tissue analyzed by enzyme-linked immunosorbent assay (ELISA) is the standard method to detect the presence of Rs in salmon sampled in hatcheries. In this study, we tested the validity of using ELISA on kidney tissue collected from intact carcasses recovered on the spawning grounds to monitor BKD in naturally spawning populations by comparing ELISA optical density (OD) values from kidney tissue that was subjected to conditions that simulated decomposition in a carcass and collection during a spawning ground survey with samples freshly collected from salmon at a hatchery. Mean ELISA OD levels were 1.060 for the samples prepared by the normal preparation and 1.115 for samples prepared by simulating spawning ground survey collection. There was no significant difference in mean ELISA OD between the 2 sample preparations and the relationship did not significantly differ from 1:1 (slope = 0.946). This demonstrates that BKD prevalence in natural populations can be monitored using ELISA conducted on samples from intact carcasses recovered on spawning ground surveys. This will be an important tool for monitoring the effect of hatchery supplementation on naturally spawning salmon populations.     

Baltic salmon, Salmo salar, from Swedish river Lule älv is more resistant to furunculosis compared to rainbow trout

Background

Furunculosis, caused by Aeromonas salmonicida, continues to be a major health problem for the growing salmonid aquaculture. Despite effective vaccination programs regular outbreaks occur at the fish farms calling for repeated antibiotic treatment. We hypothesized that a difference in natural susceptibility to this disease might exist between Baltic salmon and the widely used rainbow trout.

Study Design

A cohabitation challenge model was applied to investigate the relative susceptibility to infection with A. salmonicida in rainbow trout and Baltic salmon. The course of infection was monitored daily over a 30-day period post challenge and the results were summarized in mortality curves.

Results

A. salmonicida was recovered from mortalities during the entire test period. At day 30 the survival was 6.2% and 34.0% for rainbow trout and Baltic salmon, respectively. Significant differences in susceptibility to A. salmonicida were demonstrated between the two salmonids and hazard ratio estimation between rainbow trout and Baltic salmon showed a 3.36 higher risk of dying from the infection in the former.

Conclusion

The finding that Baltic salmon carries a high level of natural resistance to furunculosis might raise new possibilities for salmonid aquaculture in terms of minimizing disease outbreaks and the use of antibiotics.     

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.     

Chararacteristics of Vagococcus salmoninarum isolated from diseased salmonid fish

Isolates of the salmonid pathogen Vagococcus salmoninarum were recovered from Atlantic salmon, rainbow trout and brown trout with peritonitis. The phenotypes of these isolates and the type strain of Vag. salmoninarum NCFB 2777 were determined by morphological, biochemical and physiological tests and whole cell protein profiles by SDS-PAGE. There was a high level of phenetic similarity between the salmonid isolates and the type strain. The species forms short Gram-positive rods, hydrolyses L-pyrrolidonyl-β-naphthylamide, is α-haemolytic on sheep's blood agar, grows at pH 9·6 and 10°C but not at 40°C or in 6·5% NaCl and is catalase-negative; a Lancefield group N antigen is not present. Vagococcus salmoninarum can be distinguished phenetically from similar fish pathogens including Carnobacterium piscicola, Enterococcus seriolicida and Lactococcus piscium.     

The viscosity and glycoprotein biochemistry of salmonid mucus varies with species,salinity and the presence of amoebic gill disease

Fish mucus has previously been reported to change in appearance and composition among species and in response to changes in salinity and disease status. This study reports on the mucus viscosity and glycoprotein biochemistry of Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum) in freshwater and seawater, both naïve to and affected by amoebic gill disease (AGD). Cutaneous mucus viscosity was measured over a range of shear rates (11.5, 23, 46 and 115 s^–1), and non-Newtonian behaviour was demonstrated for all three species. Mucus viscosity was significantly greater in seawater than in freshwater for all species, and significantly lower in AGD-affected Atlantic salmon and brown trout. Mucus glucose, total protein and osmolality data indicated that differences in viscosity due to salinity were mostly attributed to changes in mucus hydration, while differences due to disease were mostly attributed to changes in mucus composition. Trends in gill mucus cell histochemistry included shifts in glycoproteins from neutral mucins in freshwater to acidic mucins in seawater, and shifts towards neutral mucins, with an increase in mucus cell numbers, in response to AGD. Results suggested that Atlantic salmon and brown trout are more similar to one another in their mucus profile than to rainbow trout. Atlantic salmon and brown trout both exhibited a whole-body mucus response to AGD, whereas rainbow trout exhibited only a local gill response. Findings hold implications for fish physiology and pathology, and indicate that future fish-disease management strategies should be species and condition specific.Communicated by I.D. HumeThe word mucus has been used in its noun form throughout the paper for clarity

---

An erratum to this article can be found at .     

Prevalence and analysis of Renibacterium salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha in North Puget Sound

Renibacterium salmoninarum causes bacterial kidney disease (BKD), a chronic and sometimes fatal disease of salmon and trout that could lower fitness in populations with high prevalences of infection. Prevalence of R. salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha inhabiting neritic marine habitats in North Puget Sound, Washington, USA, was assessed in 2002 and 2003. Fish were collected by monthly surface trawl at 32 sites within 4 bays, and kidney infections were detected by a quantitative fluorescent antibody technique (qFAT). The sensitivity of the qFAT was within an order of magnitude of the quantitative real-time PCR (qPCR) sensitivity. Prevalence of infection was classified by fish origin (marked/hatchery vs. unmarked/likely natural spawn), month of capture, capture location and stock origin. The highest percentages of infected fish (63.5 to 63.8%) and the greatest infection severity were observed for fish collected in Bellingham Bay. The lowest percentages were found in Skagit Bay (11.4 to 13.5%); however, there was no difference in prevalence between marked and unmarked fish among the capture locations. The optimal logistic regression model of infection probabilities identified the capture location of Bellingham Bay as the strongest effect, and analysis of coded wire tagged (CWT) fish revealed that prevalence of infection was associated with the capture location and not with the originating stock. These results suggest that infections can occur during the early marine life stages of Chinook salmon that may be due to common reservoirs of infection or horizontal transmission among fish stocks.     

Seasonality and heterogeneity of live fish movements in Scottish fish farms

Movement of live animals is a key contributor to disease spread. Farmed Atlantic salmon Salmo salar, rainbow trout Onchorynchus mykiss and brown/sea trout Salmo trutta are initially raised in freshwater (FW) farms; all the salmon and some of the trout are subsequently moved to seawater (SW) farms. Frequently, fish are moved between farms during their FW stage and sometimes during their SW stage. Seasonality and differences in contact patterns across production phases have been shown to influence the course of an epidemic in livestock; however, these parameters have not been included in previous network models studying disease transmission in salmonids. In Scotland, farmers are required to register fish movements onto and off their farms; these records were used in the present study to investigate seasonality and heterogeneity of movements for each production phase separately for farmed salmon, rainbow trout and brown/sea trout. Salmon FW-FW and FW-SW movements showed a higher degree of heterogeneity in number of contacts and different seasonal patterns compared with SW-SW movements. FW-FW movements peaked from May to July and FW-SW movements peaked from March to April and from October to November. Salmon SW-SW movements occurred more consistently over the year and showed fewer connections and number of repeated connections between farms. Therefore, the salmon SW-SW network might be treated as homogeneous regarding the number of connections between farms and without seasonality. However, seasonality and production phase should be included in simulation models concerning FW-FW and FW-SW movements specifically. The number of rainbow trout FW-FW and brown/sea trout FW-FW movements were different from random. However, movements from other production phases were too low to discern a seasonal pattern or differences in contact pattern.     

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha,O. kisutch,O. mykiss) but not by salinity

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.     

Tissue astaxanthin and canthaxanthin distribution in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

A comparative investigation of tissue carotenoid distribution between rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar, was undertaken to identify the relative efficiency of utilization of astaxanthin and canthaxanthin. Higher apparent digestibility coefficients (ADCs) (96% in trout vs. 28-31% in salmon; P<0.05), and pigment retention efficiencies (11.5-12.5% in trout vs. 5.5% in salmon; P<0.05), for both astaxanthin and canthaxanthin, were observed for rainbow trout. Astaxanthin deposition was higher than canthaxanthin in rainbow trout, while the reverse was true for Atlantic salmon, suggesting species-specificity in carotenoid utilization. The white muscle (95% in trout vs. 93% in salmon) and kidneys (0.5% in trout vs. 0.2% in salmon) represented higher proportions of the total body carotenoid pool in rainbow trout than in Atlantic salmon (P<0.05), whereas the liver was a more important storage organ in Atlantic salmon (2-6% in salmon vs. 0.2% in trout; P<0.05). The liver and kidney appeared to be important sites of carotenoid catabolism based on the relative proportion of the peak chromatogram of the fed carotenoid in both species, with the pyloric caecae and hind gut being more important in Atlantic salmon than in the rainbow trout. Liver catabolism is suspected to be a critical determinant in carotenoid clearance, with higher catabolism expected in Atlantic salmon than in rainbow trout.     

Body temperature and seawater adaptation in farmed Atlantic salmon and rainbow trout during prolonged chilling

The core temperature of the rainbow trout Oncorhynchus mykiss (3·5 kg) dropped to 1·0° C during the first 6 h of chilling at 0·5° C, remained stable until 24 h, and dropped significantly to 0·7° C after 39 h. Blood plasma osmolality increased and muscle moisture content decreased gradually with increasing chilling time. After 39 h of chilling, the rainbow trout experienced 40 mosmol l^-1 higher blood plasma osmolality and 2·8% less muscle moisture content compared with initial values. In the Atlantic salmon Salmo salar (5·3 kg), core temperature dropped to 1·3° C and blood plasma osmolality increased significantly during the first 6 h of chilling at 0·5° C, but remained relatively stable throughout the rest of the experimental period. After 39 h of chilling, the salmon experienced 20 mosmol l^-1 higher blood plasma osmolality and 0·5% less muscle moisture content compared with initial values. In rainbow trout muscle moisture content was inversely related to blood plasma osmolality indicating reduced seawater adaptation with increasing hours of chilling. No such relationship was observed in the Atlantic salmon. Hence, changes in plasma osmolality and muscle moisture in the Atlantic salmon do not indicate osmoregulatory failure since the new levels, once established, were maintained throughout the chilling time.     

Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes

Kidney and spleen homogenates from each of 60 coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri) were examined for detection of Renibacterium salmoninarum. The proportions of positives differed widely with the detection procedures used: in coho salmon, 5% were positive by the Gram-stain procedure, 10% by the direct fluorescent antibody test, 48% by bacteriological isolation, 65% by staphylococcal coagglutination, and 73% by counterimmunoelectrophoresis; in steelhead trout, 3% were positive by Gram-stain, 8.3% by fluorescent antibody, 17% by bacteriological isolation, and 67% by counterimmunoelectrophoresis. Renibacterium salmoninarum was not detected in either coho salmon or steelhead trout by immunodiffusion analysis.     

Susceptibility of rainbow trout Oncorhynchus mykiss,Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.     

A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha

The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37 degrees C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57- and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.